Phenotypic divergence and overlap between leukemic cells in elderly AML vs their normal maturation-stage counterpart in healthy adults. (A) Bone marrow samples from older AML patients (N = 265) and healthy adults (N = 30; age range, 20-24 years) were immunophenotyped with the first 5 combinations of the EuroFlow panel for the diagnostic classification of MDS/AML. Patient-specific aberrant phenotypes were identified and the total leukemic cells were exported into new FCS files without the remaining nucleated cells. Files from patients in whom ≥50% leukemic cells expressed CD34 were merged with files containing CD34⁺ progenitors from healthy adults; files from patients whom <50% leukemic cells expressed CD34 but ≥50% expressed CD117, were merged with files containing CD117⁺ myeloid or erythroid progenitors from healthy adults, depending on the lineage commitment of leukemic cells; files from patients whom <50% leukemic cells expressed CD34 and CD117 were merged with files containing the total neutrophil, monocytic, or erythroid lineage from healthy adults, depending on the lineage commitment of leukemic cells. After merging FCS files of leukemic cells from patients and the corresponding normal-cell counterpart from healthy adults, cells were plotted using principal component analysis (PCA) and represented according to their median value of expression for any given 8 markers in the combination (leukemic cells) or to the standard deviation (SD) (normal counterpart). In each combination, a score of −1, 0, or 1 was given if leukemic cells were plotted inside, over or outside the SD of the normal counterpart. Thus, patients could be scored from −5 (full phenotypic overlap with the normal counterpart) to 5 (full phenotypic divergence with respect to the normal counterpart). (B) Number of patients with each score based on the principal component analysis of merged data.